Targeting Of Multiple Myeloma By Pomalidomide Combined T Lymphocyte Redirected With A Novel CD138-specific Chimeric Antigen Receptor

Multiple myeloma is the proliferation of abnormal plasma cells, eventually leading to monoclonal immunoglobulin abnormal accumulation of B-lineage malignancies. Because of the aging of population and progress of diagnose, the incidence is climbing, and the most commonly used chemotherapy and stem cell transplantation were unable to achieve a cure, and subject to the patient’s age, organ function and other factors. So we need to find another therapeutic method which is more safe、effective、targetable and low side effect. Immunotherapy is an ideal therapeutic method. Although the appearing of the first generation immunomodulatory agent thalidomide, the second generation immunomodulatory agent lenalidomide and proteasome inhibitor bortezomib improved clinical outcomes in patients with MM, but with the extension of the course, the majority of patients disease eventually relapse. It is the third generation immunomodulator which developed by the company of Celgene. February 8,2013, the US Food and Drug Administration (FAD) approved Pomalidomide used for the treatment of relapsed, refractory multiple myeloma patients. Pomalidomide can reduce cytokines such as interleukin-6, tumor necrosis factor-a which can promote tumor cell survival, down the key function signals of MM cells. Immune dysfunction is characteristic of MM, MM patients had varying degrees of immune surveillance and response defects. It can directly stimulate T lymphocytes, increase the secretion of IFN-γ and other Thl-type cytokines. Foreign clinical studies suggest that, Pomalidomide joint dexamethasone treatment of recurrent, refractory patients can make some patients in remission, prolong survival. Compared to the first generation and second-generation immunomodulatory agents, its pharmacological effect is stronger, better tolerated, less toxic and safer. But there is still a lack of effective treatment in some patients for clinical response, existing treatment is very limited basis, the rapid development of modern cancer immunotherapy brings hope for cancer patients. T lymphocytes plays a major role in tumor immune response, but the tumor can escape identify and kill through a variety of mechanisms, and inhibit T cell function. T lymphocytes based adoptive immunotherapy has made some clinical effect, but not very satisfactory. Chimeric antigen receptor is a transmembrane protein synthesis, after genetic modification, it can target to the tumor cells, compared with previous adoptive T cell immunotherapy, the biggest advantage of CAR-T cells is unlimited by MHC. Our study is to be intended to transducer T cells with a lentiviral vector encoding a chimeric antigen receptor, the genetically modified T cells stably expressing the chimeric antigen receptor specific for CD 138, which is highly expressed on myeloma cells surface, can induce intensified anti-myeloma immune response, and reinforce the apoptosis of myeloma cells. T cells can be easily expanded to large numbers and readily available on demand in standardized quality for adoptive therapy. In this study, different concentrations of pomalidomide acting on MM cell lines RPMI8226, U266,to investigate the effect in vitro on RPMI8226, U266, and further observe the apoptosis of CD138-CAR-T, pomalidomide combines CD138-CAR-T cells on RPMI8226, U266 in vitro. Experimental study includes the following two parts:Part ⅠEffect of Pomalidomide on human Multiple Myeloma cells RPMI8226 and U266Objective:This study investigated the effects of pomalidomide (CC-4047) on the proliferation, apoptosis of human multiple myeloma (MM) cells U266 cell and RPMI8226 cell, the change of apoptosis signal protein Caspase-8、IRF-4 and CRBN mRNA expression.Methods:The two type of MM cells were reacted with pomalidomide separately and cellular morphology change was observed using light microscope; apoptosis rate, cell cycle and expression change of NF-kB were analyzed with flow cytometer; the mRNA expression level change of IRF-4, Caspase-8 and CRBN were examined by RT-PCR.Result:Pomalidomide did not shown significant effects on the cellular proliferation, cell cycle, morphology and NF-kB expression level in RPMI8226 cell (P>0.05). While for U266 cells, Pomalidomide arrested the cell cycle in G0/G1 phase and induced cellular apoptosis in a dosage-dependent manner (P<0.05); the mRNA expression level of IRF-4 and CRBN were decreased. Caspase-8 was up regulation in U266 cells (P<0.05).Conclusion:It is concluded that Pomalidomide is capable of induce U266 cell apoptosis, and arrest cell cycle in GO/G1 phase, whereas show no effect to RPMI8226 cell.Part ⅡThe cytotoxity of Pomalidomide combined CAR-T for multiple myeloma cell RPMI8226 and U266Objective:This study aimed to investigate the cytotoxity of CD138-CAR-T cells on human multiple myeloma cell RPMI8226 and U266 cells,to explore the effect of pomalidomide on CD138-CAR-T cells and the cytotoxity of CD138-CAR-T on RPMI8226 and U266 cells.Methods:The cytotoxity of CD138-CAR-T and CD138-CAR-T combined pomalidomide on RPMI8226 and U266 was detected by CFSE/7AAD. The cells were co-cultured with target cells at 5:1, after incubation for 18h, the supernatant were collected and used for ELISA assays.Results:After 18h, the cytotoxity of CD138-CAR-T on RPMI8226 and U266 was significantly higher than control(P<0.01). There was no significantly change on the cytotoxity of pomalidoide combined with CD138-CAR-T on RPMI8226 and U266.The result showed that co-cultured system contributed to a markedly increased production of IFN-γ, after adding pomalidomide to the co-cultured system, it can significantly enhanced the production of IFN-γ, compared with CD138-CAR-T.Conclusion:CD138-CAR-T had significantly cytotoxity on U266 and RPMI8226, co-culture can promote the secretion of IFN-γ. Pomalidomide can promote CD138-CAR-T cells secrete IFN-γ.